Potentials and limitations of Integra® flowable wound matrix seeded with adipose tissue-derived microvascular fragments

Abstract

Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularisation units for bioengineered Integra ® matrix wound dressing (MWD). However, due to the sheet-like structure with small pore sizes, the seeding of this matrix with ad-MVF is mainly limited to its surface. Integra ® flowable wound matrix (FWM) may be suitable to achieve a more homogeneous distribution and, thus, improved vascularisation, because this gel-like matrix allows for the direct admixture of ad-MVF during sample preparation. To test this hypothesis, we seeded MWD and FWM with an identical number of ad-MVF and assessed their distribution and inter-fragment distance within both matrices. Moreover, ad-MVF-seeded MWD and FWM were implanted into full-thickness skin defects within mouse dorsal skinfold chambers to analyse their vascularisation, epithelialisation and tissue incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Seeded FWM exhibited a more homogeneous ad-MVF distribution, when compared to MWD. This resulted in a significantly increased interfragment distance, preventing the reassembly of ad-MVF into new microvascular networks. Accordingly, the vascularisation of FWM was diminished after implantation, as indicated by a reduced functional microvessel density and blood perfusion. This was associated with a decreased tissue incorporation and epithelialisation of the matrix, when compared to ad-MVF-seeded MWD. Hence, the use of FWM as a carrier system may require a tremendous amount of ad-MVF to shorten their inter-fragment distance and, thus, to maintain their vascularisation capacity for tissue engineering applications.

Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments

“…Using intravital fluorescence microscopy, we found that HD and MD ad-MVF-seeded Integra ® exhibited a comparable vascularisation over time, whereas LD ad-MVF-seeded implants were characterised by a significantly lower number of perfused ROIs and functional microvessel density. In line with these findings, additional photoacoustic measurements (Später et al, 2017). This process not only involves the interconnection of individual ad-MVF with each other but also the development of anastomoses with the surrounding vessels of the host tissue.…”

“…The vascularisation of the implants was assessed in 12 regions of interest (ROIs). ROIs exhibiting red blood cell (RBC)-perfused microvessels were defined and counted as perfused ROIs (in % of all ROIs) (Später et al, 2017). The functional microvessel density was determined as the total length of all RBC-perfused microvessels per ROI (cm/cm 2 ).…”

“…For this purpose, angiogenic sprouts, growing out of the vessel www.ecmjournal.org T Später et al ad-MVF density affects implant vascularisation segments, have to develop interconnections (Nunes et al, 2010). Since the growth rate of such angiogenic sprouts is ~5 µm/h (Utzinger et al, 2015), the distance between individual ad-MVF crucially determines the kinetics of this network formation process (Später et al, 2017). These considerations suggest that a high seeding density of ad-MVF markedly contributes to the success of this vascularisation strategy.…”

Prevascularization of dermal substitutes with adipose tissue-derived microvascular fragments enhances early skin grafting

“…Moreover, >95% of the microvessels within the prevascularized implants were GFP + , indicating that they originated from the seeded ad-MVF. This finding is important, because in contrast to previous studies 11 , 30 , 31 we herein used the double-layer Integra, which is covered with a silicone sheet. Hence, ad-MVF could only be seeded on the bottom side of the matrices and were implanted in direct contact to the bone of the mouse skull.…”

Enoxaparin does not affect network formation of adipose tissue–derived microvascular fragments

“…Under experimental conditions, ad-MVF have already been shown to improve the vascularization of random-pattern flaps, 31 superficial myocardium, 32 epicardial patches, 33 volumetric muscle defects, 34 pancreatic encapsulating devices, 35 and scaffolds for tissue engineering. 36 A major prerequisite to pave the way for the transfer of such applications into clinical practice is to evaluate whether the vascularization capacity of ad-MVF is affected by clinical routine procedures. In this context, we could herein demonstrate that enox does not impair the reassembly of ad-MVF into new microvascular networks within implanted dermal skin substitutes.…”